Filter unit for filtering and method for collecting cells in a liquid cell culture medium

ABSTRACT

A liquid cell culture medium collecting filter unit includes a porous metal membrane that filters out cells in a liquid cell culture medium, a support that holds a peripheral portion of the porous metal membrane. and a tubular member that has a hollow part serving a flow path for a liquid cell culture medium. The tubular member is connected to the support such that the flow path faces at least part of a main surface of the porous metal membrane.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of application Ser. No.16/408,829, filed May 10, 2019, now U.S. Pat. No. 11,091,730, which is acontinuation of International application No. PCT/JP2017/038065, filedOct. 20, 2017, which claims priority to Japanese Patent Application No.2016-225227, filed Nov. 18, 2016, the entire contents of each of whichare incorporated herein by reference.

BACKGROUND OF THE INVENTION

The present invention relates to a liquid cell culture medium collectingfilter unit, a liquid cell culture medium collecting method, and aliquid cell culture medium collecting kit.

Cell (cultured cell) growth requires a culture medium serving as anutrient source. The culture medium needs to be replaced at regularintervals. A known method for replacing a culture medium uses, forexample, centrifugation (see Japanese Unexamined Patent ApplicationPublication No. 2004-105139, hereinafter Patent Document 1).

The method for replacing a culture medium using centrifugation iscarried out, for example, as follows.

First, a cell-containing liquid cell culture medium in a containertargeted for culture medium replacement is dispensed into centrifugationtubes. Next, each centrifugation tube is centrifuged, so that cells areconcentrated at the bottom of each centrifugation tube. Next, thesupernatant, which is part of the liquid cell culture medium, in eachcentrifugation tube is collected. Next, the cell-containing liquid cellculture medium that remains in each centrifugation tube is returned tothe container. Next, a fresh liquid cell culture medium is placed in thecontainer. Accordingly, replacement of the culture medium in thecontainer is completed.

In the method for replacing culture media using centrifugation, the stepof collecting the supernatant which is part of the liquid cell culturemedium is performed by humans. Therefore, some workers may collect notonly the supernatant but also cells and reduce the proportion of cellsremaining in the container. In other words, there are large variationsin the proportion of remaining cells depending on worker. In addition,it takes a long time to collect the liquid cell culture medium.

To solve the above-described issues, it is an object of the presentinvention is to provide a liquid cell culture medium collecting filterunit, a liquid cell culture medium collecting method, and a liquid cellculture medium collecting kit, which can reduce variations in theproportion of remaining cells and can shorten the time required tocollect a liquid cell culture medium.

BRIEF SUMMARY OF THE INVENTION

A liquid cell culture medium collecting filter unit according to anaspect of the invention comprises a porous metal membrane that filtersout cells in a liquid cell culture medium, a support that holds aperipheral portion of the porous metal membrane, and a tubular memberthat has a hollow part serving as a flow path for the liquid cellculture medium. The tubular member is connected to the support such thatthe flow path faces at least part of a main surface of the porous metalmembrane.

The support preferably includes first and second frame members betweenwhich the peripheral portion of the porous metal membrane is sandwiched.The peripheral portion of the porous metal membrane preferably has asurface located between first and second spaced bent portions that issandwiched between the first and second frame members. The surfacepreferably has at least one stripe-shaped protrusion located between onthe surface. More preferably, the surface has a plurality of thestripe-shaped protrusions located between the first and second bentportions and the stripe-shaped protrusions are oriented in randomdirections.

The porous metal membrane is preferably flush or substantially flushwith an opening plane defined by an end portion of the protrusion of theholding member. As used herein, the term “substantially flush” meansthat the gap between S2 and element 2 of FIG. 4 (as measured in thevertical direction is FIG. 4 ) is 400 μm or less.

As annular protrusion that protrudes in a thickness direction of thesupport is preferably provided on a main surface of the support andextends away from the tubular member. In such a case, the porous metalmembrane is preferably disposed on an inner side of the annularprotrusion.

A liquid cell culture medium collecting method according to an aspect ofthe present invention includes the following acts:

placing a liquid cell culture medium collecting filter unit in acontainer that contains a cell-containing liquid cell culture medium,the liquid culture medium collecting filter unit including a porousmetal membrane that filters out cells in the liquid cell culture medium,a support that holds a peripheral portion of the porous metal membrane,and a tubular member that has a hollow part serving as a flow path forthe liquid cell culture medium and is connected to the support such thatthe flow path faces at least part of a main surface of the porous metalmembrane; and

passing the liquid cell culture medium in the container through the flowpath and the porous metal membrane and collecting the liquid cellculture medium outside the container.

The liquid cell culture medium collecting method preferably furtherincludes supplying a fresh liquid cell culture medium to the containerafter at least some of the liquid cell culture medium has been collectedoutside the container. It is useful to pass the liquid cell culturemedium located in the container passes through the porous metal membranein a first direction as it is being moved from the container to alocation outside the container and to pass the fresh liquid cell culturemedium through the porous metal membrane in a second direction, oppositeto the first direction, as it is supplied to the container.

After the fresh liquid cell culture medium has been supplied to thecontainer supplying, it is preferable to cause at least some of theliquid cell culture medium located in the container to flow through theflow path and the porous metal membrane and to collect the liquid cellculture medium outside the container.

A liquid cell culture medium collecting kit according to another aspectof the invention comprising the liquid cell culture medium collectingfilter unit described above.

The liquid cell culture medium collecting filter unit and the liquidcell culture medium collecting method according to the aspects of thepresent invention can reduce variations in the proportion of remainingcells and can shorten the time required to collect a liquid cell culturemedium.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view illustrating the schematic structure of aliquid cell culture medium collecting filter unit according to anembodiment of the present invention.

FIG. 2 is an exploded perspective view of the liquid cell culture mediumcollecting filter unit in FIG. 1 .

FIG. 3 is an exploded sectional view of the liquid cell culture mediumcollecting filter unit in FIG. 1 .

FIG. 4 is an assembly sectional view of the liquid cell culture mediumcollecting filter unit in FIG. 1 .

FIG. 5 is a partially enlarged sectional view of FIG. 4 .

FIG. 6 is a partially enlarged perspective view illustrating theschematic structure of a porous metal membrane.

FIG. 7 is a partially enlarged plane view illustrating the schematicstructure of a modification of the porous metal membrane.

FIG. 8 is a plane view illustrating the schematic structure of theporous metal membrane.

FIG. 9 is a schematic diagram illustrating an example of a liquid cellculture medium collecting and replacing method.

FIG. 10 is a schematic diagram illustrating a modification of the liquidcell culture medium collecting and replacing method.

FIG. 11 is a table showing the proportion of remaining cells and thereplacement time when the liquid cell culture medium in the container isreplaced by using the liquid cell culture medium collecting filter unitin FIG. 1 , where the table also shows the proportion of remaining cellsand the replacement time when the liquid cell culture medium in thecontainer is replaced by using a centrifuge in the Comparative Example.

FIG. 12A is a schematic diagram illustrating the step of replacing acell-containing liquid cell culture medium by using a centrifuge.

FIG. 12B is a schematic diagram illustrating the step following the stepin FIG. 12A.

FIG. 12C is a schematic diagram illustrating the step following the stepin FIG. 12B.

FIG. 12D is a schematic diagram illustrating the step following the stepin FIG. 12C.

FIG. 12E is a schematic diagram illustrating the step following the stepin FIG. 12D.

FIG. 12F is a schematic diagram illustrating the step following the stepin FIG. 12E.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A liquid cell culture medium collecting filter unit according to anaspect of the invention comprises a porous metal membrane that filtersout cells in a liquid cell culture medium, a support that holds aperipheral portion of the porous metal membrane, and a tubular memberthat has a hollow part serving as a flow path for the liquid cellculture medium. The tubular member is connected to the support such thatthe flow path faces at least part of a main surface of the porous metalmembrane.

Since this structure includes the porous metal membrane that filters outcells in the liquid cell culture medium, the cells can remain in thecontainer as a result of filtering out the cells by using the porousmetal membrane, and only the liquid cell culture medium can be collectedthrough liquid culture medium flow paths. This can reduce variations inthe proportion of remaining cells and achieve a high proportion ofremaining cells. Since cells are less likely to be attached to a porousmetal membrane than to, for example, a porous resin membrane, it ispossible to prevent inhibition of collection of the liquid cell culturemedium caused by clogging of membrane holes with cells. Thus, the timerequired to collect the liquid cell culture medium can be shortened.

The holding member preferably includes a first frame member and a secondframe member between which the peripheral portion of the porous metalmembrane is sandwiched. The peripheral portion of the porous metalmembrane preferably has a first bent portion and a second bent portion.The peripheral portion of the porous metal membrane is preferablysandwiched between the first frame member and the second frame member soas to have a stripe-shaped protrusion between the first bent portion andthe second bent portion. According to this structure, the stripe-shapedprotrusion between the first bent portion and the second bent portion inthe porous metal membrane can increase the friction between theperipheral portion of the porous metal membrane and the first framemember and the second frame member. This structure can avoid the porousmetal membrane from falling out of the frame members even if the liquidcell culture medium is collected at a high flow rate. The liquid cellculture medium can thus be collected in a short time.

A plurality of the stripe-shaped protrusions is preferably providedbetween the first bent portion and the second bent portion, and thestripe-shaped protrusions are preferably oriented in random directions.This structure can increase the friction between the peripheral portionof the porous metal membrane and the first frame member and the secondframe member and can further improve the holding power of the firstframe member and the second frame member used to hold the porous metalmembrane.

An annular protrusion that protrudes in the thickness direction of theholding member is preferably provided on a main surface of the holdingmember away from the tubular member. The porous metal membrane ispreferably disposed on the inner side of the protrusion. According tothis structure, the porous metal membrane can be positioned closer tothe bottom of the container, and the liquid surface can be lowered tothe main surface below the porous metal membrane. The amount of thecollected liquid cell culture medium can thus be increased withoutreducing the proportion of remaining cells. Since the porous metalmembrane positioned closer to the bottom of the container contacts alarge amount of the liquid cell culture medium, the cells attached tothe porous metal membrane are washed away, which can make it difficultto cause clogging. When a liquid cell culture medium is supplied throughthe porous metal membrane after collection of the liquid cell culturemedium, the cells attached to the porous metal membrane tend to bedetached from the porous metal membrane during the passage of the liquidcell culture medium. This can further avoid clogging. As a result, theworking time can be shortened. This configuration can also suppress anincrease in pressure in the tubular member caused by clogging and canreduce the stress on the cells.

The porous metal membrane is preferably flush or substantially flushwith an opening plane defined by the end portion of the protrusion ofthe holding member. This structure enables the porous metal membrane tobe positioned still closer to the bottom of the container and canincrease the amount of the collected liquid cell culture medium withoutreducing the proportion of remaining cells. This structure can alsoprevent the porous metal membrane from being clogged with cells toshorten the working time and can reduce the stress on the cells.

A liquid cell culture medium collecting method according to an aspect ofthe present invention includes

a filter unit-placing step of placing, in a container that contains acell-containing liquid cell culture medium, a liquid cell culture mediumcollecting filter unit including a porous metal membrane that filtersout cells in the liquid cell culture medium, a holding member that holdsthe peripheral portion of the porous metal membrane, and a tubularmember that has a hollow part serving as a flow path for the liquid cellculture medium and is connected to the holding member such that thehollow part faces at least part of a main surface of the porous metalmembrane; and

a collecting step of introducing the liquid cell culture medium in thecontainer to the flow path through the porous metal membrane andcollecting the liquid cell culture medium outside the container.

Since the liquid cell culture medium in the container is sucked throughthe porous metal membrane in this method, the cells can remain in thecontainer as a result of filtering out the cells by using the porousmetal membrane, and only the liquid cell culture medium can be collectedthrough the liquid culture medium flow paths. This configuration canreduce variations in the proportion of remaining cells. Since cells areless likely to be attached to a porous metal membrane than to, forexample, a porous resin membrane, it is possible to prevent inhibitionof collection of a liquid cell culture medium caused by clogging ofmembrane holes with cells. Thus, the time required to collect the liquidcell culture medium can be shortened.

The method may further include, after the collecting step, a supplyingstep of supplying a fresh liquid cell culture medium to the container.According to this method, the liquid cell culture medium in thecontainer can be replaced by a fresh liquid cell culture medium.

In the supplying step, the fresh liquid cell culture medium may flowthrough the flow path and may be supplied to the container through theporous metal membrane. According to this method, even if cells areattached to the porous metal membrane, the cells can be detached fromthe porous metal membrane (that is, the porous metal membrane can bebackwashed) under the pressure of the fresh liquid cell culture medium.As a result, the time required to replace the liquid cell culture mediumcan be shortened.

The method may further include, after the supplying step, are-collecting step of introducing the liquid cell culture medium in thecontainer to the flow path through the porous metal membrane andcollecting the liquid cell culture medium outside the container.According to this method, the liquid cell culture medium can becontinuously collected by using the same porous metal membrane.

A liquid cell culture medium collecting kit according to an aspect ofthe present invention is a liquid cell culture medium collecting kitused in the liquid cell culture medium collecting method and includesthe liquid cell culture medium collecting filter unit. This structurecan reduce variations in the proportion of remaining cells and canshorten the time required to collect the liquid cell culture medium.

Referring ow to the drawings wherein like numerals indicate likeelements, FIGS. 1 to 5 illustrate the structure of a liquid cell culturemedium collecting filter unit according to a preferred embodiment of thepresent invention.

As illustrated in FIG. 1 and FIG. 2 , a liquid cell culture mediumcollecting filter unit 1 includes a porous metal membrane 2, a holdingmember 3, and a tubular member 4, which is connected to the holdingmember 3. The holding member 3 holds a peripheral portion 2A of theporous metal membrane 2 as illustrated in FIGS. 3 to 5 . In thepreferred embodiment, the holding member 3 includes first and secondframe members 31 and 32.

As best shown in FIGS. 4 and 5 , the peripheral portion 2A of the porousmetal membrane 2 can be sandwiched between the first and second framemembers 31 and 32. The first frame member 31 includes an annular flatportion 31 a and an annular protrusion 31 c, which is positioned at thesection around a central through-hole 31 b so as to protrude away fromthe tubular member 4. The flat portion 31 a has a diameter of, forexample, 18 mm. The flat portion 31 a has a thickness of, for example,1.5 mm. The protrusion 31 c has a height of, for example, 1.5 mm.

An annular flange 31 d, which protrudes toward the center of thethrough-hole 31 b, is formed on the inner surface of the annularprotrusion 31 c. The flange 31 d is provided at a distance of, forexample, 0.1 mm from the top of the protrusion 31 c toward the flatportion 31 a. As best shown in FIG. 3 , an end portion 31 f of theflange 31 d close to the center of the through-hole 31 b has a reducedthickness so as to form a slope surface 31 e on the tubular member 4side. The thickness of the flange 31 d adjacent to the protrusion 31 cis, for example, 0.3 mm. The thickness of the end portion 31 f of theflange 31 d is, for example, 0.2 mm. The angle of the slope surface 31 eis, for example, 45 degrees.

As best shown in FIG. 3 , the second frame member 32 includes an annularflat portion 32 a, an annular protrusion 32 c (which is positioned atthe section around a central through-hole 32 b so as to protrude awayfrom the tubular member 4), and an annular protrusion 32 d (which ispositioned at the section around the central through-hole 32 b so as toprotrude toward the tubular member 4). The flat portion 32 a has adiameter of, for example, 18 mm. The flat portion 32 a has a thicknessof, for example, 1.5 mm. The protrusion 32 d has a height of, forexample, 1.5 mm.

The protrusion 32 c has an outer diameter that is slightly smaller thanthe diameter of the through-hole 31 b such that the protrusion 32 c canbe inserted into the through-hole 31 b of the first frame member 31. Anend portion 32 f of the protrusion 32 c is shaped in conformance withthe shape of the flange 31 d on the tubular member 4 side. In otherwords, the end portion 32 f has a slope surface 32 e corresponding tothe slope surface 31 e.

As illustrated in FIG. 4 , the porous metal membrane 2 is held so as tohave tension in the plane direction when the peripheral portion 2A issandwiched between the flange 31 d of the first frame member 31 and theend portion 32 f of the protrusion 32 c of the second frame member 32and lies along the slope surface 31 e and the slope surface 32 e. Theperipheral portion 2A of the porous metal membrane 2 is sandwiched atthe position spaced from a central plane S1 (which is a central plane inthe thickness direction of the holding member 3) in the thicknessdirection. In the preferred embodiment, the porous metal membrane 2 isdisposed on the inner side of the annular protrusion 31 c and issubstantially flush with an opening plane S2 defined by the end portionof the annular protrusion 31 c.

As illustrated in FIG. 3 , the flat portion 31 a of the first framemember 31 has a plurality of through-holes 31 g, which penetrates thefirst frame member 31 in the thickness direction. The through-holes 31 gare arranged at regular intervals in the circumferential direction ofthe flat portion 31 a. Similarly, the flat portion 32 a of the secondframe member 32 has a plurality of through-holes 32 g, which penetratesthe second frame member 32 in the thickness direction. The through-holes32 g are arranged at regular intervals in the circumferential directionof the flat portion 32 a so as to correspond to the through-holes 31 g.The first frame member 31 and the second frame member 32 are fixed toeach other by inserting fastening members (not illustrated), such asscrews, into the respective through-holes 31 g and 32 g while theprotrusion 32 c of the second frame member 32 is inserted in thethrough-hole 31 b of the first frame member 31.

The tubular member 4 includes a fitting portion 4 a, which can be fittedto the protrusion 32 d of the second frame member 32. The tubular member4 is detachably attached to the second frame member 32 by fitting thefitting portion 4 a onto the outer surface of the protrusion 32 d of thesecond frame member 32. The tubular member 4 has a hollow part 4 bserving as a flow path for the liquid cell culture medium. The tubularmember 4 is connected to the second frame member 32 such that the hollowpart 4 b faces at least part of the main surface of the porous metalmembrane 2. With this configuration, the liquid cell culture medium thathas passed through the porous metal membrane 2 can be collected throughthe hollow part 4 b which defines a flow path for the liquid cellculture medium.

Examples of the materials of the first frame member 31, the second framemember 32, and the tubular member 4 include metals, such as duralumin,aluminum, and stainless steel (SUS); and resins, such as polyethylene,polystyrene, polypropylene, polycarbonate, polyacetal, andpolyetherimide.

FIG. 6 is a partially enlarged perspective view schematicallyillustrating the structure of an exemplary porous metal membrane 2 usedto filter out cells contained in the liquid cell culture medium. Theporous metal membrane 2 is used to filter out cells (e.g., spheroids)contained in the liquid cell culture medium.

As illustrated in FIG. 6 , the porous metal membrane 2 has a pair ofopposing main surfaces 21 a and 21 b and a plurality of through-holes 21c which penetrates both the main surfaces 21 a and 21 b. Thethrough-holes 21 c separate cells from the liquid cell culture medium.The shape and size of the through-holes 21 c are appropriately setaccording to the shape and size of the cells. The through-holes 21 c maybe arranged, for example, at regular intervals or periodically. Theshape of the through-holes 21 c is, for example, square as viewed fromthe main surface 21 a side of the porous metal membrane 2. In theillustrated embodiment, the through-holes 21 c are arranged in a squarelattice. The size of each through-hole 21 c is, for example, 0.1 μm ormore and 600 μm or less in length and 0.1 μm or more and 600 μm or lessin width. The interval between the through-holes 21 c is, for example,larger than the size of the through-holes 21 c by a factor of preferably1 time or more and 10 times or less, and more preferably 3 times orless. The opening ratio of the through-holes 21 c in the porous metalmembrane 2 is, for example, 10% or more. The through holes 21 c can takeother shapes and may be, for example, regular hexagons as viewed fromthe main surface 21 a side of the porous metal membrane 2. Thethrough-holes 21 c may be arranged to form a honeycomb structure.

Examples of the material of the porous metal membrane 2 include gold,silver, copper, platinum, nickel, stainless steel, palladium, titanium,cobalt, alloys thereof, and oxides thereof. The size of the porous metalmembrane 2 is, for example, 6 mm in diameter. The thickness of theporous metal membrane 2 is, for example, 0.1 μm or more and 100 μm orless and preferably 0.1 μm or more and 50 μm or less. The porous metalmembrane 2 has, for example, a circular, elliptical, or polygonalcontour. In the preferred embodiment, the porous metal membrane 2 has acircular outer periphery. The peripheral portion of the porous metalmembrane 2 may have the through-holes 21 c or may not have thethrough-holes 21 c.

In the embodiment illustrated in FIG. 5 , the peripheral portion 2A ofthe porous metal membrane 2 is sandwiched between the slope surface 31 eof the first frame member 31 and the slope surface 32 e of the secondframe member 32 so as to have a first bent portion 2 a and a second bentportion 2 b. As illustrated in FIG. 8 , the peripheral portion 2A of theporous metal membrane 2 is sandwiched so as to have stripe-shapedprotrusions 2 c between the first bent portion 2 a and the second bentportion 2 b. The stripe-shaped protrusions 2 c refer to parts thatprotrude from one of the main surfaces of the porous metal membrane 2and have a height that is 0.1 times or more and 2 times or less thethickness of the porous metal membrane 2. The stripe-shaped protrusions2 c can increase the friction between the peripheral portion 2A of theporous metal membrane 2 and the first frame member 31 and the secondframe member 32.

As illustrated in FIG. 8 , a plurality of the stripe-shaped protrusions2 c is provided between the first bent portion 2 a and the second bentportion 2 b, and the stripe-shaped protrusions 2 c are preferablyoriented in random directions. This structure can increase the frictionbetween the peripheral portion 2A of the porous metal membrane 2 and thefirst frame member 31 and the second frame member 32 and can furtherimprove the holding power of the first frame member 31 and the secondframe member 32 used to hold the porous metal membrane 2.

The stripe-shaped protrusions 2 c may be formed of, for example, thewrinkles formed in the porous metal membrane 2. The term “wrinkles”refers to fine stripes created as a result of loosening or shrinking ofthe porous metal membrane 2. In this case, the porous metal membrane 2itself can form the stripe-shaped protrusions 2 c, and there is no needto separately dispose members serving as the stripe-shaped protrusions 2c.

Next, a liquid cell culture medium collecting and replacing method usingthe liquid cell culture medium collecting filter unit 1 according to thepreferred embodiment will be described. FIG. 9 is a schematic diagramillustrating an example of the liquid cell culture medium collecting andreplacing method.

First, as illustrated in FIG. 9 , the liquid cell culture mediumcollecting filter unit 1 is placed in a container 5 that contains aliquid cell culture medium CCS containing cells CE. More specifically,one end portion of the tubular member 4 is placed in the container 5such that the porous metal membrane 2 is positioned in the liquid cellculture medium CCS.

The other end portion of the tubular member 4 is connected to a pump P1.The pump P1 is connected to a collection pipe 61 through which theliquid cell culture medium CCS collected from the container 5 flows intoa liquid culture medium collection container 62. To supply a freshliquid cell culture medium CCS to the container 5, one end portion of asupply pipe 71 is placed in the container 5. The other end portion ofthe supply pipe 71 is connected to a pump P2. The pump P2 is connectedto a suction pipe 73 through which the fresh liquid cell culture mediumCCS is sucked from a liquid culture medium storage container 72. Thecontainer 5 has a bottle shape and has a cap 51, which closes theopening. The tubular member 4 and the supply pipe 71 penetrate the cap51.

Next, the pump P1 is driven to suck the liquid cell culture medium CCSin the container 5 through the porous metal membrane 2, and the liquidcell culture medium CCS is collected in the liquid culture mediumcollection container 62, which is located outside the container 5,through the tubular member 4. Since the porous metal membrane 2 filtersout (e.g., retains) the cells CE in the container 5 at this time, thecells CE remain in the container 5, and only the liquid cell culturemedium CCS is collected in the liquid culture medium collectioncontainer 62 through the tubular member 4 and the collection pipe 61.

Next, the pump P2 is driven to supply the fresh liquid cell culturemedium CCS in the liquid culture medium storage container 72 to thecontainer 5 through the suction pipe 73 and the supply pipe 71(supplying step). Accordingly, a fresh culture medium can be prepared byreplacing the liquid cell culture medium CCS in the container 5.

Next, after a predetermined period of time has elapsed, the pump P1 isdriven to suck the liquid cell culture medium CCS in the container 5through the porous metal membrane 2, and the liquid cell culture mediumCCS is collected in the liquid culture medium collection container 62,which is located outside the container 5, through the tubular member 4.

Next, the pump P2 is driven to supply the fresh liquid cell culturemedium CCS in the liquid culture medium storage container 72 to thecontainer 5 through the suction pipe 73 and the supply pipe 71.Accordingly, the fresh culture medium can be prepared by replacing theliquid cell culture medium CCS in the container 5 again.

Since the liquid cell culture medium collecting filter unit 1 includesthe porous metal membrane 2 according to the preferred embodiment, thecells CE can remain in the container 5 as a result of filtering out thecells CE by using the porous metal membrane 2, and only the liquid cellculture medium CCS can be collected through the liquid culture mediumflow paths (the tubular member 4 and the collection pipe 61). Thisconfiguration can reduce variations in the proportion of cells CEremaining in the container 5.

In the preferred embodiment, since the cells CE are less likely to beattached to the porous metal membrane 2 than to, for example, a porousresin membrane, it is possible to prevent inhibition of collection ofthe liquid cell culture medium CCS caused by clogging of membrane holeswith the cells CE. Thus, the time required to collect the liquid cellculture medium CCS can be shortened.

Since the porous metal membrane 2 has higher mechanical stiffness than,for example, a porous resin membrane or the like, the porous metalmembrane 2 is unlikely to change in size even when always immersed inthe liquid cell culture medium CCS or even under high pressure.Therefore, the collection and replacement of the liquid cell culturemedium can be automated and repeated by controlling the drive of thepumps P1 and P2.

According to the preferred embodiment, there is no need to remove thecap 51 from the container 5 at the time of the collection andreplacement of the liquid cell culture medium. This makes it possible toobtain a substantially sealed space in the container 5 so as to preventcontamination by bacteria and the like.

According to the preferred embodiment, the holding member 3 includes thefirst frame member 31 and the second frame member 32 between which theperipheral portion 2A of the porous metal membrane 2 is sandwiched. Theperipheral portion 2A of the porous metal membrane 2 has the first bentportion 2 a and the second bent portion 2 b. The peripheral portion 2Aof the porous metal membrane 2 is sandwiched between the first framemember 31 and the second frame member 32 so as to have the stripe-shapedprotrusions 2 c between the first bent portion 2 a and the second bentportion 2 b. According to this structure, the stripe-shaped protrusions2 c between the first bent portion 2 a and the second bent portion 2 bin the porous metal membrane 2 can increase the friction between theperipheral portion 2A of the porous metal membrane 2 and the first framemember 31 and the second frame member 32. This structure can avoid theporous metal membrane 2 from falling out of the position between thefirst frame member 31 and the second frame member 32 even if the liquidcell culture medium CCS is collected at a high flow rate. The liquidcell culture medium CCS can thus be collected in a short time.

According to the preferred embodiment, a plurality of the stripe-shapedprotrusions 2 c is provided between the first bent portion 2 a and thesecond bent portion 2 b, and the stripe-shaped protrusions 2 c areoriented in random directions. This structure can increase the frictionbetween the peripheral portion 2A of the porous metal membrane 2 and thefirst frame member 31 and the second frame member 32 and can furtherimprove the holding power of the first frame member 31 and the secondframe member 32 used to hold the porous metal membrane 2.

According to the preferred embodiment, the annular protrusion 31 c isprovided on a main surface 3 a of the holding member 3 and projects awayfrom the tubular member 4. The porous metal membrane 2 is disposed onthe inner side of the protrusion 31 c. This structure enables the porousmetal membrane 2 to be positioned closer to the bottom of the container5 and can increase the amount of the collected liquid cell culturemedium CCS without reducing the proportion of remaining cells CE. Theliquid surface can be lowered to the main surface below the porous metalmembrane 2. The amount of the collected liquid cell culture medium CCScan thus be increased without reducing the proportion of remaining cellsCE. Since the porous metal membrane 2 positioned closer to the bottom ofthe container 5 contacts a large amount of the liquid cell culturemedium, the cells attached to the porous metal membrane 2 are washedaway, which can make it difficult to cause clogging. When a liquid cellculture medium CCS is supplied through the porous metal membrane 2 aftercollection of the liquid cell culture medium, the cells CE attached tothe porous metal membrane 2 tend to be detached from the porous metalmembrane 2 during the passage of the liquid cell culture medium CCS.This can further avoid clogging. As a result, the working time can beshortened. This configuration can also suppress an increase in pressurein the tubular member 4 caused by clogging and can reduce the stress onthe cells.

According to the preferred embodiment, the porous metal membrane 2 isflush or substantially flush with an opening plane defined by the endportion 31 f of the protrusion 31 c of the holding member 3. The porousmetal membrane 2 can be positioned still closer to the bottom of thecontainer 5, and the amount of the collected liquid cell culture mediumCCS can be increased without reducing the proportion of remaining cellsCE. The porous metal membrane 2 can be prevented from being clogged withcells to shorten the working time and to reduce the stress on the cells.

According to the preferred embodiment, the collecting step is followedby the supplying step, and thus the liquid cell culture medium in thecontainer 5 can thus be replaced by a fresh liquid cell culture medium.

According to the preferred embodiment, the supplying step is followed bythe re-collecting step, and thus the liquid cell culture medium CCS canbe continuously collected by using the same porous metal membrane 2.

The present invention is not limited to the above-described embodimentand can be carried out in various other aspects. For example, in theforegoing description, the fitting portion 4 a of the tubular member 4is fitted onto the protrusion 32 d of the second frame member 32,whereby the tubular member 4 is attached to the second frame member 32.The present invention is not limited to this configuration. For example,one of the fitting portion 4 a of the tubular member 4 and theprotrusion 32 d of the second frame member 32 may have an externalthread, and the other may have an internal thread, such that the tubularmember 4 and the second frame member 32 can be screwed to each other.Alternatively, for example, the second frame member 32 may have alocking claw (e.g., a hook-shaped claw), and the tubular member 4 mayhave a receptacle (e.g., a hole engageable with the hook-shaped claw),such that the locking claw can be fitted into the receptacle.

In the foregoing description, the pump P1 is driven to suck the liquidcell culture medium CCS in the container 5 through the porous metalmembrane 2, and the liquid cell culture medium CCS is collected in theliquid culture medium collection container 62, which is located outsidethe container 5, through the tubular member 4. The present invention isnot limited to this configuration. Alternatively, the liquid cellculture medium CCS in the container 5 may be introduced to the tubularmember 4 through the porous metal membrane 2 and collected outside thecontainer 5. For example, the liquid cell culture medium CCS in thecontainer 5 is not sucked into the tubular member 4 but pressure-fed tothe tubular member 4 through the porous metal membrane 2.

In the foregoing description, the fresh liquid cell culture medium CCSin the liquid culture medium storage container 72 is supplied to thecontainer 5 through the supply pipe 71 in the supplying step. Thepresent invention is not limited to this configuration. For example, asillustrated in FIG. 10 , the other end portion of the tubular member 4may be branched, and the branches of the branched tubular member may berespectively connected to the pumps P1 and P2. According to thisstructure, the fresh liquid cell culture medium CCS in the liquidculture medium storage container 72 can flow through the tubular member4 and can be supplied to the container 5 through the porous metalmembrane 2. As a result, even if the cells CE are attached to the porousmetal membrane 2, the cells CE can easily be detached from the porousmetal membrane 2 (that is, the porous metal membrane can easily bebackwashed) under the pressure of the fresh liquid cell culture mediumCCS, and the time required to replace the liquid cell culture medium CCScan be shortened. In this case, the tubular member 4 is preferablyprovided with valves V1 and V2 as illustrated in FIG. 10 to prevent theliquid cell culture medium CCS collected from the container 5 from beingmixed with the fresh liquid cell culture medium CCS.

In the foregoing description, the container 5 has a bottle shape. Thepresent invention is not limited to this shape. By way of example, andnot limitation, the container 5 may be a container like a beaker or maybe a resin container like an intravenous drip bag. The container 5 isany container that can contain a liquid cell culture medium.

In FIG. 9 and FIG. 10 , the liquid culture medium collection container62 and the liquid culture medium storage container 72 are beakers. Thepresent invention is not limited to beakers. Like the container 5, theliquid culture medium collection container 62 and the liquid culturemedium storage container 72 may have a bottle shape and have a cap thatcloses the opening. According to this structure, the liquid cell culturemedium CCS can be collected and replaced while the containers are sealedsuch that the cells CE and the liquid cell culture medium CCS are out ofcontact with outside air. As a result, the possibility of contaminationof the cells CE and the liquid cell culture medium CCS can be reduced.

The liquid cell culture medium collecting kit for use in the liquid cellculture medium collecting method according to the preferred embodimentincludes the liquid cell culture medium collecting filter unit 1. Theliquid cell culture medium collecting kit may further include thecontainer 5.

Next, the results of experiments that were carried out to study the timerequired to replace the liquid cell culture medium CCS in the container5 and the proportion of cells CE remaining in the container 5 will bedescribed. FIG. 11 is a table showing the results of those experiments.More particularly, it shows, inter alia, the proportion of cells CEremaining in the container 5 after the culture medium CCS has beensucked out of the container 5 and placed in the container 62 and thereplacement time when the liquid cell culture medium in the container 5is replaced using the liquid cell culture medium collecting filter unit1 according to the preferred embodiment. FIG. 11 also shows theproportion of remaining cells CE and the replacement time when theliquid cell culture medium CCS in the container is replaced by using acentrifuge in a Comparative Example.

In this experiment, ras gene-introduced NIN3T3 cells were cultured byusing a cell culture multiwell plate (available from Sumitomo BakeliteCo., Ltd.) with 96 wells and a U-shaped well bottom to produce 270 ormore spheroids. The culture time was controlled so as to producespheroids having a diameter of about 600 μm. The spheroids thus producedas the cells CE were added to the liquid cell culture medium CCS. Theliquid cell culture medium CCS was a Dulbecco's modified Eagle's mediumDMEM (High Glucose, Nacalai tesque, Inc.: 08458-45) containing 1% apenicillin-streptomycin mixed solution and 5% fetal bovine serum. Aplurality of containers 5 that each contained 200 mL of a liquid cellculture medium CCS containing 30 spheroids were prepared, and aplurality of containers 5 that each contained 200 mL of a liquid cellculture medium CCS containing 60 spheroids were prepared.

As the porous metal membrane 2, a porous metal membrane having thestructure illustrated in FIG. 7 (hole size (distance between opposingsides of hexagon): 100 μm, line width: 13 μm, opening ratio: 78%,honeycomb structure) and a porous metal membrane having the structureillustrated in FIG. 6 (hole size (length of sides of square): 200 μm,line width: 75 μm, opening ratio: 53%, square lattice) were used.Through the porous metal membrane, 190 mL of the liquid cell culturemedium CCS (200 mL) containing either 30 or 60 spheroids was sucked fromthe container 5 (see FIG. 9 ) and deposited in container 62. At thistime, the output of the pump P1 was 100 mL/min. A fresh liquid cellculture medium CCS was the supplied to the container 5 from the liquidculture medium storage container 72 until the total amount of the liquidcell culture medium CCS in the container 5 reached 200 mL. The liquidcell culture medium CCS in the container 5 was then filtered through theporous metal membrane (hole size: 50 μm), and the cells CE remaining onthe porous metal membrane were observed under a microscope. The resultsare shown in FIG. 11 .

As illustrated in FIG. 11 , replacement of the liquid cell culturemedium CCS containing 30 spheroids using a liquid cell culture mediumcollecting filter 1 unit having the porous metal membrane 2 (hole size:100 μm) was performed twice, and the proportion of the cells CEremaining in the container 5 after each replacement operation was 86.7%and 63.3%, respectively. The two replacement operations (the operationof moving the liquid cell culture medium CCS from the culture mediumstorage container 72 to the container 5) was 2 minutes and 46 seconds,and 2 minutes and 35 seconds, respectively. In separate experiments, thereplacement operations were performed by replacing the liquid cellculture medium CCS containing 60 spheroids using a liquid cell culturemedium collecting filter unit having the porous metal membrane (holesize: 100 μm) was performed twice, and the proportion of the cells CEremaining in the container 5 after the replacement operation was 90.0%and 88.3%, respectively. The replacement work time was 2 minutes and 52seconds, and 2 minutes and 41 seconds.

In a similar manner, the replacement of the liquid cell culture mediumCCS containing 30 spheroids using a liquid cell culture mediumcollecting filter unit having the porous metal membrane (hole size: 200μm) was performed twice. The proportion of the cells CE remaining in thecontainer 5 after the two replacement operations was 90% and 96.7%,respectively. The time it took to carry out the replacement operationswere 2 minutes and 45 seconds, and 2 minutes and 37 seconds,respectively. In another experiment, a liquid cell culture medium CCScontaining 60 spheroids was placed in the container 5 and by using aliquid cell culture medium collecting filter unit having the porousmetal membrane (hole size: 200 μm) was performed twice, and theproportion of the cells CE remaining in the container 5 was 75.0% and96.7%. The time it took to replace the liquid cell culture medium CCSwas 3 minutes and 00 seconds, and 2 minutes and 42 seconds,respectively.

In the Comparative Example, the experiment illustrated in FIG. 12A toFIG. 12F was performed by using a centrifuge.

First, a plurality of containers 5A that each contain 200 mL of a liquidcell culture medium CCS containing 30 or 60 spheroids was prepared as inthe foregoing description.

Next, as illustrated in FIG. 12A, 40-mL aliquots of the liquid cellculture medium CCS in the container 5A were dispensed into fivecentrifugation tubes 101.

Next, as illustrated in FIG. 12B, the centrifugal force was applied tothe centrifugation tubes 101 by using a centrifuge 102 at a rotationspeed of 1000 rpm for 5 minutes.

Next, as illustrated in FIG. 12C, the supernatant which is part of theliquid cell culture medium CCS in each centrifugation tube 101 wasdiscarded into a waste container 103.

Next, as illustrated in FIG. 12D, the cell CE-containing liquid cellculture medium CCS that remains in each centrifugation tube 101 wasreturned to the container 5A.

Next, as illustrated in FIG. 12E, 10 mL of a fresh liquid cell culturemedium CCS was placed in each centrifugation tube 101, and the inside ofeach centrifugation tube 101 was washed with the fresh liquid cellculture medium CCS. The liquid cell culture medium CCS obtained afterwashing was placed in the container 5A.

Next, as illustrated in FIG. 12F, the fresh liquid cell culture mediumCCS was placed in the container 5A such that the total amount of theliquid cell culture medium CCS in the container 5A reached 200 mL. Theliquid cell culture medium CCS in the container 5A was filtered throughthe porous metal membrane (hole size: 50 μm), and the cells CE remainingon the porous metal membrane were observed under a microscope. Theresults are shown in FIG. 11 .

As illustrated in FIG. 11 , the work of replacing the liquid cellculture medium CCS containing 30 spheroids by using the centrifuge 102was performed twice, and the proportion of the cells CE remaining in thecontainer 5A was 53.3% and 30.0%. The replacement work time was 22minutes and 12 seconds, and 17 minutes and 53 seconds. The work ofreplacing the liquid cell culture medium CCS containing 60 spheroids byusing the centrifuge 102 was performed twice, and the proportion of thecells CE remaining in the container 5A was 90.0% and 96.3%. Thereplacement work time was 21 minutes and 44 seconds, and 17 minutes and14 seconds.

The experimental results indicate that the liquid cell culture mediumcollecting filter unit 1 according to the preferred embodiment canreduce variations in the proportion of the remaining cells CE (63.5% to96.7%→30.0% to 96.3%). The experimental results also indicate that theliquid cell culture medium collecting filter unit 1 according to thepreferred embodiment can greatly shorten the time required to collectthe liquid cell culture medium CCS.

In the field of cell culture, a liquid culture medium containingsecretes from cells may be used. The liquid culture medium is preparedby repeating the process of immersing cells in a liquid culture mediumfor a certain period of time, then collecting only the cells, andimmersing the collected cells again for a certain period of time. Inpreparing the liquid culture medium, a large amount of the liquidculture medium can be prepared by using the liquid cell culture mediumcollecting filter unit (hereinafter abbreviated as the filter unit) 1according to the preferred embodiment.

The results of the experiment in which a liquid culture medium isprepared by using the filter unit 1 according to the preferredembodiment will be described below.

First, a conditioned medium was prepared by adding 1 liter of aserum-free medium containing 105 CHO-RD cells/mL to a 1-liter culturecontainer. The filter unit 1 according to the preferred embodiment wasplaced in the culture container. The tubular member 4 was a siliconehose (6 mm in inner diameter). The porous metal membrane 2 was a porousmetal membrane (diameter of exposed portion: 6 mm, thickness: 1.2 μm,hole size (length of sides of square): 2.6 μm, line diameter: 1.0 μm,opening ratio: 52%, square lattice) made of nickel and having thestructure illustrated in FIG. 6 . A tubing pump was connected to thetubular member 4 at about the midpoint of the tubular member 4. One endportion of the tubular member 4 located outside the culture containerwas placed in a 1-liter closed collection container. In this state, theconditioned medium was left to stand for two days to culture the cellsin the culture container.

The tubing pump was then operated to transfer 0.9 liters of theconditioned medium from the culture container to the collectioncontainer. The operating time of the tubing pump at this time was 30minutes.

The filter unit 1 was then taken out of the culture container, and 100mL of the liquid remaining in the culture container was taken out. Thenumber of the cells in the liquid was determined with a cell counter.

As a result, the cell concentration in the liquid was 107 cells/mL. Thesurface of the filter unit 1 taken out of the culture container wasobserved under a microscope and a small number of the cells were presenton the surface.

The results of the experiment in which a liquid culture medium isprepared using the filter unit according to Comparative Example will bedescribed below.

The filter unit according to the Comparative Example was different fromthe filter unit 1 according to the preferred embodiment in that a porousresin membrane (hole size (diameter of circular hole): 3.0 μm, openingratio: 20%, random arrangement) was used instead of the porous metalmembrane 2. Otherwise, the filter unit according to the ComparativeExample had the same structure as the filter unit 1 according to thepreferred.

First, the filter unit according to the Comparative Example was placedin a culture container containing the conditioned medium, and one endportion of the tubular member located outside the culture container wasplaced in a 1-liter closed collection container. In this state, theconditioned medium was left to stand for two days to culture the cellsin the culture container.

The tubing pump was then operated to transfer 0.9 liters of theconditioned medium from the culture container to the collectioncontainer. The operating time of the tubing pump at this time was 120minutes.

The filter unit according to the Comparative Example was then taken outof the culture container, and 100 mL of the liquid remaining in theculture container was taken out. The number of the cells in the liquidwas determined with a cell counter.

As a result, the cell concentration in the liquid was 5×104 cells/mL. Asthe surface of the filter unit according to the Comparative Exampletaken out of the culture container was observed under a microscope, alarge number of the cells were present on the surface.

The above-described experimental results indicate that the cellconcentration of the liquid remaining in the culture container in thecase of using the filter unit 1 according to the preferred embodiment is200 times higher than that in the case of using the filter unitaccording to the Comparative Example. In other words, the use of thefilter unit 1 according to the preferred embodiment enables continuouspreparation of the liquid culture medium and enables preparation of alarger amount of the liquid culture medium. Accordingly, the filter unit1 is particularly effective when rare cells are used to prepare liquidculture media.

One of the reasons why the cell concentration of the liquid remaining inthe culture container in the case of using the filter unit 1 accordingto the preferred embodiment is higher than that in the case of using thefilter unit according to the Comparative Example may be because theporous metal membrane always contacts a large amount of the liquid cellculture medium, and the cells attached to the porous metal membrane arewashed away, which makes it difficult to cause clogging. Another reasonmay be because the pressure required to transfer the liquid culturemedium in the case of using the filter unit 1 according to the preferredembodiment is lower than that in the case of using the filter unitaccording to the Comparative Example, which reduces clogging caused bythe cells. In particular, when the porous membrane is disposed in thehollow part of the tubular member, the pressure loss caused by thetubular member as well as the pressure loss caused by the porousmembrane occur. In the filter unit according to the Comparative Exampleincluding the porous resin membrane, the pressure in the tubular membermay become extremely high at the time of occurrence of clogging, and theclogging caused by the cells may become more severe. Thus, the filterunit in Example can shorten the work time and can reduce the stress onthe cells. When a porous metal membrane is used as in the filter unit 1according to the preferred embodiment, the cells attached to the porousmetal membrane may easily be released into the liquid at the time ofstopping of suction of the liquid culture medium, and the reduction inthe number of cells in the culture container may be reduced.

Although the present invention is fully described in connection withpreferred embodiments and with reference to the accompanying drawings,various modifications and alterations will be apparent to those skilledin the art. It should be understood that these modifications andalterations are within the scope of the present invention defined by theaccompanying claims unless the modifications and alterations are out ofthe scope of the present invention.

Since the liquid cell culture medium collecting filter unit according tothe present invention can reduce variations in the proportion ofremaining cells and can shorten the time required to collect a liquidcell culture medium, the liquid cell culture medium collecting filterunit according to the present invention is useful to collect and replacethe liquid cell culture medium.

REFERENCE SIGNS LIST

1 Liquid cell culture medium collecting filter unit

2 Porous metal membrane

2A Peripheral portion

2 a First bent portion

2 b Second bent portion

2 c Stripe-shaped protrusion

3 Holding member

3 a, 3 b Main surface

4 Tubular member

4 a Fitting portion

4 b Hollow part

5, 5A Container

21 a, 21 b Main surface

21 c Through-hole

31 First Frame Member

31 a Flat portion

31 b Through-hole

31 c Protrusion

31 d Flange

31 e Slope surface

31 f End portion

31 g Through-hole

32 Second frame member

32 a Flat portion

32 b Through-hole

32 c Protrusion

32 d Protrusion

32 e Slope surface

32 f End portion

32 g Through-hole

51 Cap

61 Collection pipe

62 Liquid culture medium collection container

71 Supply pipe

72 Liquid culture medium storage container

73 Suction pipe

101 Centrifugation tube

102 Centrifuge

103 Waste container

P1, P2 Pump

The invention claimed is:
 1. A filtering unit for filtering cellscontained in a liquid cell culture medium, the filter comprising:tubular member defining a liquid cell culture medium flow path having acentral axis; a porous membrane comprising a central portion located inthe flow path and a peripheral portion located outside of the flow path;and a support member holding the peripheral portion of the porousmembrane in such a manner that the central portion of the porousmembrane is in tension and lies in a plane which is perpendicular to thecentral axis, the support member including: a first frame member having:a first protrusion extending parallel to the central axis and away fromthe porous membrane; and a flange extending toward the central axis andbeing radially inward of the first protrusion; and a second frame membercooperating with the first frame member to hold the peripheral portionof the porous membrane, the second frame member having: a secondprotrusion extending parallel to the central axis and toward the porousmembrane, the first and second protrusions cooperating to hold theperipheral portion of the porous membrane; and a third protrusionextending parallel to the central axis and being connected to thetubular member.
 2. The filtering unit according to claim 1, wherein thefirst protrusion is cylindrical in shape.
 3. The filtering unitaccording to claim 2, wherein the second protrusion is cylindrical inshape.
 4. The filtering unit according to claim 3, wherein the thirdprotrusion is cylindrical in shape.
 5. The filtering unit according toclaim 1, wherein the support member has first and second opposedsurfaces that extend at an oblique angle with respect to the centralaxis and hold the peripheral portion of the porous membrane such thatthe peripheral portion of the porous membrane is sandwiched between thefirst and second opposed surfaces.
 6. The filtering unit according toclaim 5, wherein a portion of the peripheral portion that is sandwichedby the first and second opposed surfaces has at least one stripe-shapedprotrusion located therein.
 7. The filtering unit according to claim 5,wherein a portion of the peripheral portion that is sandwiched by thefirst and second opposed surfaces has at a plurality of stripe-shapedprotrusion located therein.
 8. The filtering unit according to claim 1,wherein the porous membrane is a metallic.
 9. The filtering unitaccording to claim 1, wherein third protrusion extends away from theporous membrane into the tubular member.
 10. The filtering unitaccording to claim 1, wherein the flange has a circular shape as viewedalong a plane extending parallel to the central axis.
 11. The filteringunit according to claim 1, wherein the second protrusion is radiallyinward of the third protrusion.
 12. The filtering unit according toclaim 1, wherein the flow path is defined by the combination of thetubular member and the support member.
 13. A liquid cell culture mediumcollecting method, comprising: placing a liquid cell culture mediumcollecting filter unit in a container that contains a cell-containingliquid cell culture medium, the liquid culture medium collecting filterunit including: a tubular member defining a liquid cell culture mediumflow path having a central axis; a porous metal membrane that filtersout cells in the liquid cell culture medium, the porous metal membranehaving a central portion located in the flow path and a peripheralportion located outside of the flow path; and a support member holdingthe peripheral portion of the porous metal membrane in such a mannerthat the central portion of the membrane is in tension and lies in aplane which is perpendicular to the central axis, the support memberincluding: a first frame member having: a first protrusion extendingparallel to the central axis and away from the porous membrane; and aflange extending toward the central axis and being radially inward ofthe first protrusion; and a second frame member cooperating with thefirst frame member to hold the peripheral portion of the porousmembrane, the second frame member having: a second protrusion extendingparallel to the central axis and toward the porous membrane, the firstand second protrusions cooperating to hold the peripheral portion of theporous membrane; and a third protrusion extending parallel to thecentral axis and being connected to the tubular member; and passing theliquid cell culture medium in the container through the flow path andthe porous metal membrane and collecting the liquid cell culture mediumoutside the container.
 14. The liquid cell culture medium collectingmethod according to claim 13, further comprising supplying a freshliquid cell culture medium to the container after at least some of theliquid cell culture medium has been collected outside the container. 15.The liquid cell culture medium collecting method according to claim 14,wherein: the liquid cell culture medium in the container passes throughthe porous membrane in a first direction as it is being moved from thecontainer to a location outside the container; and the fresh liquid cellculture medium passes through the porous membrane in a second direction,opposite to the first direction, as it is supplied to the container. 16.The liquid cell culture medium collecting method according to claim 15,further comprising, after the fresh liquid cell culture medium has beensupplied to the container, causing at least some of the liquid cellculture medium located in the container to flow through the flow pathand the porous membrane and collecting the liquid cell culture mediumoutside the container.
 17. The liquid cell culture medium collectingmethod according to claim 14, further comprising, after the fresh liquidcell culture medium has been supplied to the container, causing at leastsome of the liquid cell culture medium located in the container to flowthrough the flow path and the porous membrane and collecting the liquidcell culture medium outside the container.
 18. The liquid cell culturemedium collecting method according to claim 13, wherein the supportmember has first and second opposed surfaces that extend at an obliqueangle with respect to the central axis and hold the peripheral portionof the porous membrane such that the peripheral portion of the porousmembrane is sandwiched between the first and second opposed surfaces.19. The liquid cell culture medium collecting method according to claim13, wherein the porous membrane is a metallic membrane.